【作者】 徐琳；

【导师】 韦革宏；

【作者基本信息】 西北农林科技大学 ， 微生物学， 2013， 博士

【摘要】 根瘤菌的固氮作用是生物固氮中最高效的体系，在维持生态平衡和农业可持续发展中发挥着重要作用。为了探讨采自祁连山不同海拔两种隶属于蝶形花亚科的野生豆科植物根瘤共生体的多样性以及生物地理因素对根瘤内生菌种群分布的影响，我们对祁连山部分地区豆科植物进行了根瘤菌资源的调查，从中选出了分别位于高、低海拔的两种优势豆科物种（箭叶锦鸡儿和黄花棘豆），并对其进行了多样性及系统发育研究。16S rDNA PCR-RFLP结果分析表明，供试菌株3种酶酶切组合形成了35个类型，对每种类型选取代表菌株进行16S rDNA全序列测序。16S rDNA全序列系统发育分析显示，供试菌株分属于根瘤菌属（Rhizobium）、中华根瘤菌属（Sinorhizobium）、叶杆菌属（Phyllobacterium）、副球菌属（Paracoccus）、微枝形杆菌属（Microvirga）、鞘脂单胞菌属（Sphingomonas）、分枝杆菌属（Mycobacterium）、类芽孢杆菌属（Paenibacillus）、科赫氏菌属（Cohnella）、芽孢八叠球菌属（Sporosarcina）、芽孢杆菌属（Bacillus）、葡萄球菌属（Staphylococcus）、短杆菌属（Brevibacterium）、色诺菲吕斯菌属（Xenophilus）、欧文氏菌属（Erwinia）、勒克菌属（Leclercia）、不动杆菌属（Acinetobacter）和假单胞菌属（Pseudomonas）的分支。它们分别归属于变形菌门、放线菌门和厚壁菌门。其中108个归属于α-变形菌纲，11个属于β-变形菌纲；25个属于γ-变形菌纲。56个属于厚壁菌门中的Paenibacillus、Brevibacillus、Staphylococcus、Sporosarcina、Cohnella和Bacillus。1个属于放线菌门中的Mycobacterium。其中根瘤菌Rhizobium、Sinorhizobium和Phyllobacterium有100株，优势种类为Rhizobium，占到71株，在系统发育上分属于Rhizobium radiobacter、Rhizobium massiliae及Rhizobium sp.分支，说明在野生豆科植物中Rhizobium存在着占优势数量的土壤杆菌。内生菌中占优势的是Bacillus、Acinetobacter和Xenophilus，分别占到了33、16和11株。另外，有八个类群（Rhizobium、Microvirga、Mycobacterium、Cohnella、 Paenibacillus、Sporosarcina、Bacillus和Xenophilus）菌株的16S rDNA序列与已知所有菌株的序列相似性小于97%，可能为潜在的新种。ERIC指纹图谱有82种类型，在一些RFLP一致的菌株间存在着不同的ERIC图谱，说明和RFLP相比，REIC指纹图谱有更高的种间、种内分辨率，同时揭示了供试菌中在种水平上丰富的多样性。在交叉结瘤试验中，仅有34株菌株与其原宿主结瘤，不和其他经典结瘤试验推荐种结瘤。nifH及nodA系统发育研究发现，16S rDNA中处于β-变形菌纲的Xenophilus和厚壁菌门的Acinetobacter都得到了有效扩增条带，测序结果表明，它们分别位于Mesorhizobium和Sinorhizobium的发育分支。说明nodA基因和nifH基因可能在根瘤菌与内生菌间发生了水平转移。生物地理学分析显示，海拔和宿主植物的类型对内生菌的基因型分布起到了主要影响。植物促生长试验表明供试菌株中大部分能够分泌IAA和产生铁载体，对五种病原菌的拮抗试验显示，除了极少数对苹果炭疽病和小麦赤霉病有抑制作用外，大多数菌株都具有对西瓜枯萎病，黄瓜炭疽病和苹果罗纹病的抑制作用。从黄花棘豆根瘤中分离出四株好氧、无芽孢、能运动的革兰氏阴性杆菌，并进行了多相分类研究。16S rRNA序列显示，它们属于Rhizobium属的未知新分支。除一株和其它三株有一个碱基差异外，其它三株有相同的16S rRNA序列，且其模式菌株CCNWQLS01与种内已知种Rhizobium oryzae的最大相似性为94.4%。这一结果被其他两个持家基因（recA和glnA）的全序列分析、表型特征测试及共生特性试验等试验所证实。其模式株CCNWQLS01的醌型以辅酶Q-10为主，脂肪酸主要类型为成分8（C18:1ω7c/C18:1ω6c;67.2%），符合典型根瘤菌的特征。基于以上研究，这四株菌形成了一个不同于现有Rhizobium属所有已知种的新种。我们将其命名为Rhizobium qilianshanensesp. nov.，菌株CCNWQLS01T（=ACCC05747~T=JCM18337~T）为其模式株。更多还原

【Abstract】 The association between legume plants and rhizobia is quite old and enables the plant tosurvive nutrient poor conditions because of the nitrogen fixation capacity of the rhizobia thatreside inside their root nodules. Legumes are a large family which occurs almost everywhereon earth and is divided into three subfamilies, the Caesalpinioideae, Mimosoideae andFaboideae. In China most legumes occur from this last subfamily. The aim of this study wasto make an in-depth analysis from the diversity of root nodule endosymbionts present in twoindigenous legumes (Caragana jubata and Oxytropis ochrocephala) in Qilian Mountains.A total of201endophytic bacteria associated to root noudules collected from twoindigenous legumes in different altitude of Qilian Mountains (Hexi Corridor) werecharacterized by16S rDNA polymerase chain reaction (PCR)-restriction fragment lengthpolymorphism,16S rRNA gene sequence analysis and enterobacterial repetitive intergenicconsensus-PCR clustering. Phylogenetically, these isolates belonged to35species in thegenera Phyllobacterium, Ensifer, Rhizobium, Microvirga, Sphingomonas, Paracoccus,Mycobacterium, Paenibacillus, Cohnella, Sporosarcina, Bacillus, Staphylococcus,Brevibacterium, Xenophilus, Erwinia, Leclercia, Acinetobacter and Pseudomonas. Thisrevealed the presence of on the one hand traditional rhizobia (50%), Phyllobacterium,Sinorhizobium and Rhizobium and on the other hand non-rhizobial endophytes belonging toseveral genera in the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria,Actinobacteria, Firmibacteria and Sphingobacteria. The non-rhizobial endophyte populationwas dominated by Bacillus, Xenophilus and Acinetobacter species. Besides, eight new groupswith sequence similarity below97%were found within the genera Rhizobium, Microvirga,Mycobacterium, Cohnella, Paenibacillus, Sporosarcina, Bacillus and Xenophilus.The symbiosis genes nodA and nifH were investigated for a selection of strains whichrevealed the presence of different symbiovars in traditional rhizobia including Sophoraalopecuroides, Medicago sativa, Phaseolus vulgaris, Pisum sativum, Vigna unguiculata,Trifolium repens, Glycyrrhiza max and Galega oficinalis. As a result, only thirty-four stainscould form nodules with the host plants. Additionally, Phylogenetic sequence analysis ofnodA showed higher similarity with those of Sinorhizobium meliloti within strains related tothe genera Rhizobium, Acinetobacter and Sinorhizobium. NifH sequences revealed strains belonged to Rhizobium, Acinetobacter, Phyllobacterium and Sinorhizobium had similar nifHgene with Sinorhizobium. Two representive strains belong to Xenophilus and Acinetobacterformed a novel nifH type, which distinct from any previously described symbiovar withinMesorhizobium. This indicate that horizontal gene transfer could have occurred betweenrhizobia and non-rhizobial endophytes. Moreover, the elevation and the host plant types didcontribute to the separation of the bacterial endosymbionts. PGP test showed most strainshave the ability of production of IAA, siderophore and antifungal activity.Four strains isolated from nodules of Oxytropis ochrocephala were classified in thegenus Rhizobium on the basis of their16S rRNA gene sequences. These strains have identical16S rRNA gene sequences, except for one strain with one base-pair different from the others.Their sequences showed a mean similarity of94.4%with the most closely related species,Rhizobium oryzae. Analysis of recA and glnA sequences showed that these strains have lessthan88.1%and88.7%similarity with the defined species of Rhizobium, respectively. Thegenetic diversity revealed by ERCI-PCR fingerprinting indicated that the isolates correspondto different strains. Strain CCNWQLS01Tcontained Q-10as the predominant ubiquinone. Themajor fatty acids were feature8(C18:1ω7c and/or C18:1ω6c;67.2%), Therefore, a novelspecies Rhizobium qilianshanense sp. nov. is proposed, and CCNWQLS01T(=ACCC05747~T=JCM18337~T) is designated as the type strain.This study provided valuable information about the interactions among the symbioticbacteria, nonsymbiotic bacteria and their habitats, thus increased the knowledge about theirgenetic diversity and ecology.更多还原